Novel iron compositions and methods of making and using the same

Abstract

To provide an aqueous iron sucrose composition having desirable properties, and a method for preventing or treating renal disease or disorder comprising administering the composition.SOLUTION: The present invention involves a novel aqueous iron composition. The aqueous iron composition includes iron sucrose and bicarbonate. The aqueous iron composition of the invention exhibits enhanced renal protective effects relative to conventional iron sucrose compositions.SELECTED DRAWING: Figure 22

Description

Technical Field

[0001] This application claims the benefit of priority of Provisional Application No. 62 / 812,028, filed on February 28, 2019, entitled "Novel Iron Compositions and Methods of Marking and Using the Same", which is hereby incorporated by reference in its entirety.

Background Art

[0002] Intravenous (IV) iron agents are colloids consisting of spherical iron-carbohydrate nanoparticles. Each particle has an iron oxyhydroxide gel core, which is surrounded by a carbohydrate shell that stabilizes the iron oxyhydroxide (the main function of the ligand is to stabilize the complex and protect it from further polynuclearization).

[0003] Since the iron carbohydrate complex must release iron from the iron (III) hydroxide core, it acts as a prodrug. According to the proposed mechanism, after administration, stable (type 1) complexes such as carboxymaltose ferric iron and iron dextran are taken up by endocytosis by macrophages of the reticuloendothelial system (RES). See Danielson, J. Structure, chemistry, and pharmacokinetics of intravenous iron agents. Am. Soc. Nephrol. 2004, 15, S93-S98.

[0004] In the case of iron(III)-carbohydrate (type 2) with low stability, especially when administered at high doses, a significant amount of unstable iron is released from the complex, leading to saturation of transferrin and thus potentially resulting in a significant amount of non-transferrin-bound iron (NTBI). This weakly bound Fe3+ can be readily taken up in a manner not regulated by cells and can induce oxidative stress. Evans, R.W.; Rafique, R.; Zarea, A.; Rapisarda, C.; Cammack, R.; Evans, P.J.; Porter, J.B.; Hider, R.C. Nature of non-transferrin-bound iron: studies on iron citrate complexes and the thalassemic era. J. Biol. Inorg. Chem. 2008, 13, 57-74.

[0005] There are currently five injectable iron-carbohydrate products approved by the FDA(1) INFeD(R) / Dexferrum(R) (iron dextran), Ferahem(R) (ferumoxytol), Injectafer(R) (iron carboxymaltose), Venofer(R) (iron sucrose), Ferrlecit(R) (sodium ferric gluconate complex). Iron sucrose, sold under the name Venofer(R), is formulated as a colloidal suspension with a molecular weight (M w ) of approximately 34,000 - 60,000 daltons and the following molecular formula.

[0006] [Na 2 Fe 5 O 8 (OH)·3(H2O)] n m(C 12 H 22 O 11 ) where n is the degree of iron polymerization and m is the number of sucrose molecules (C 12 H 22 O 11 ) forming a complex with the polynuclear polymeric iron core.

[0007] [Na2 Fe 5 O 8 (OH)·3(H2O)] n Each mL contains 20 mg of elemental iron as iron sucrose in water for injection. Venofer(R) is available in 5 mL single-dose vials (100 mg of elemental iron per 5 mL) and 10 mL single-dose vials (200 mg of elemental iron per 10 mL). The formulation contains approximately 30% sucrose w / v (300 mg / mL) and has a pH of 10.5 - 11.1. The product does not contain preservatives. The osmolarity of the injection is 1,250 mOsmol / L.

[0008] A method for synthesizing iron carbohydrates is described in Lawrence et al.'s WO97 / 11711 (1997), which discloses an iron(III) oxyhydroxide-dextran composition for treating iron deficiency having ellipsoidal particles with a preferred molecular weight range of about 250,000 - 300,000 daltons.

[0009] In recent years, iron sucrose has been used in combination with tin protoporphyrin (SnPP) to induce acquired cellular resistance without causing damage to organs. See U.S. Patent No. 9,844,563 to Zager et al.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0011]

Non-Patent Document 1

Non - Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0012] The inventors have found a need for an iron sucrose formulation for its renal protective effect that can be easily combined with tin protoporphyrin (SnPP), is stable, can be injected into patients for treating iron deficiency, or can be used alone or in combination with another agent such as SnPP.

Means for Solving the Problems

[0013] The present invention relates to an aqueous iron sucrose composition having desirable properties. In one aspect, the aqueous iron sucrose composition contains iron sucrose and bicarbonate. In one aspect, the present invention relates to a pharmaceutical aqueous iron pharmaceutical composition containing iron sucrose; bicarbonate; and a pharmaceutically acceptable aqueous carrier. In another aspect, the present invention relates to a method for preventing or treating a kidney disease or disorder, which includes intravenously administering an aqueous iron composition containing iron sucrose and bicarbonate in a therapeutically effective amount.

Brief Description of the Drawings

[0014]

Figure 1

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Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Mode for Carrying Out the Invention

[0015] In one embodiment, the present invention includes an aqueous iron sucrose (FeS) and bicarbonate (FeS - bicarbonate) composition. The inventors have found that this composition has beneficial properties. In one aspect, the FeS - bicarbonate composition of the present invention can be utilized as a renal protective agent. The inventors have discovered that the FeS - bicarbonate composition according to the embodiments of the present invention is preferentially absorbed by the kidney as compared to commercially available forms of FeS. Furthermore, the inventors have found that FeS - bicarbonate results in a preferential up - regulation of renal protective molecules as compared to FeS alone. In another aspect, the FeS - bicarbonate composition of the present invention can be advantageously combined with other renal protective agents, such as tin protoporphyrin (SnPP), to readily form an injectable renal protective agent.

[0016] One advantage of using FeS - bicarbonate is that this composition brings about an increase in the renal protective effect. Specifically, the inventors have found that FeS - bicarbonate preferentially up - regulates renal protective molecules as compared to FeS alone. Without wishing to be bound by theory, the inventors have proposed that in addition to FeS, bicarbonate can change the relative levels of existing Fe(III) and Fe(II). Since a reddish color of the FeS - bicarbonate product was observed, the inventors have proposed that the composition of the present invention may contain a high level of Fe(II). Considering that Fe(II) is more reactive than Fe(III), this may explain the increase in the renal protective effect.

[0017]

[0017] One advantage of using FeS-bicarbonate is that the bicarbonate has a buffering effect. This can be advantageous when using a tin protoporphyrin composition, as SnPP is best stored at low pH to prevent unwanted dimerization during storage. According to the present disclosure, the SnPP composition can be combined with the FeS-bicarbonate composition at an SnPP:FeS ratio of about 1:1 or less, such as about 1:2, about 1:4, about 1:8, about 1:10, about 1:20, about 1:50, about 1:100, about 1:1000, about 1:10,000, about 1:100,000, about 1:1,000,000, or any integer or subrange therebetween.

[0018] In one aspect, the composition has a molecular weight measured using the GPC described in Example 1. Mp is preferably in the range of 25,000 to 35,000 Daltons, more preferably in the range of 28,000 to 32,000 Daltons, and most preferably about 29,000 Daltons. Mw is preferably in the range of 25,000 to 45,000 Daltons, more preferably in the range of 30,000 to 40,000 Daltons, even more preferably in the range of 33,000 to 38,000 Daltons, and most preferably about 34,000 Daltons. Mn is preferably in the range of 15,000 to 30,000 Daltons, more preferably in the range of 20,000 to 25,000 Daltons, and most preferably about 24,000 Daltons. The polydispersity index (PDI) is preferably in the range of 1.35 to 1.60, more preferably in the range of 1.38 to 1.5, even more preferably in the range of 1.40 to 1.48, and most preferably about 1.4.

[0019]

[0019] In one aspect, the composition has a stable zeta potential of -3.0 mV or less, more preferably -7.0 mV or less, and most preferably about -10 mV. In one aspect, the composition has less than 8.5% total organic carbon, preferably less than 8.0%, and most preferably about 7.7%. In one aspect, the weight osmolality measured according to Example 1 is in the range of 550 to 1600 mOsm / kg, preferably in the range of 1500 to 1580 mOsm / kg, and most preferably about 1540 mOsm / kg.

Example

[0020] [Example 1] The present invention includes a composition prepared by dissolving an iron sucrose complex sufficient to obtain a solution of 12 mg / mL (expressed as iron) in water (about 3.5 L) when diluted to 6.0 L. The amount of iron sucrose required is calculated for a final liquid volume of 6100 mL (6.1 L), and the final concentration is 12 mg / mL. This requires 73.2 g of iron. The potency of the iron sucrose used is 0.0550. Therefore, 73.2 g / 0.0550 or 1331 g ± 1 g of iron sucrose is required. 1331 g ± 1 g of iron sucrose was weighed directly into a 6.0 L Erlenmeyer flask. About 3 - 3.5 L of water was added to the Erlenmeyer flask and the contents of the flask were stirred.

[0021] Sodium bicarbonate is added in an amount such that the final sodium bicarbonate concentration is 10 mg / mL when diluted to 6.0 L. 109.8 ± 0.1 g of sodium bicarbonate was weighed and added to a 6.0 L flask.

[0022] Sodium chloride is added in an amount such that the final sodium chloride concentration is 9.0 mg / mL upon dilution. (54.9 ± 0.1) of sodium chloride was weighed and added to a 6.0 L flask. The suspension was stirred for 30 - 120 minutes to obtain a black opaque solution.

[0023] Small amounts of 1 M sodium hydroxide were added until a stable pH of 10.30 was reached, while monitoring the pH of the solution with a pH meter. 40.0 ± 0.1 g of sodium hydroxide was added to a 1.0 L Erlenmeyer flask. 1.0 ± 0.1 L of water was added to the 1.0 L Erlenmeyer flask and stirred until all the sodium hydroxide was dissolved. A pH probe was fixed to monitor the pH of the 6.0 L Erlenmeyer flask, and sodium hydroxide was added in <100 mL portions until pH = 10.3 ± 0.1. The solution was then stirred for 10 minutes. After 10 minutes, the pH was checked again and adjusted to within pH = 10.3 ± 0.1 if necessary.

[0024] Next, transfer the solution into a volumetrically accurate flask and dilute it to 6.1 L with water. Use a 2 L volumetric flask twice to accurately transfer 4 L of the 10.3 pH solution into a 6 L Erlenmeyer flask. Dilute the remaining 10.3 pH solution to 2 L with a volumetric flask and add it to the 6 L Erlenmeyer flask. Using a 100 mL graduated cylinder, add 100 ± 0.1 mL to the 6.0 L Erlenmeyer flask and stir the resulting solution for 10 minutes.

[0025] The resulting product solution appears dark red to brown. Two isotopes of iron are present in the sample preparation in a ratio that matches the ratio of a standard preparation. The pH of the resulting material is 10.3, which is within the preferred range of 10.1 - 10.4. The resulting material had 11.5 / 11.6 parts per thousand (mg / mL) of iron according to SOP174472 for determining iron by inductively coupled plasma mass spectrometry.

[0026] Further properties of the resulting composition are shown in Table 1 below.

[0027] [Table 1] TIFF2025084949000003.tif229169

[0028] The resulting FeS - bicarbonate composition has the following stoichiometry and physical constants are shown in Table 2 below:

[0029] [Table 2]

[0030] [Example 2] When intravenous administration of the iron sucrose (FeS) bicarbonate composition of Example 1 was carried out for 4 hours, renal heme oxygenase 1 (HO - 1) increased compared to a commercially available iron sucrose (FeS) composition sold under the trade name Venofer(R). The results are shown in Table 3 below.

[0031]

Table 3

[0032] The elevated levels of HO-1 observed in the kidney were not observed in the liver. Instead, no increase in the levels of HO-1 was observed with FeS-bicarbonate as compared to that observed with Venofer(R). The results are shown in Table 4 below.

[0033]

Table 4

[0034] Plasma BUN and creatinine were similar for both FeS, Venofer(R), and FeS-bicarbonate as shown in Tables 5 and 6 below.

[0035]

Table 5

[0036]

Table 6

[0037] [Example 3] The FeS-bicarbonate composition of Example 1 was filtered and placed into vials and had an FeS concentration of 12 mg / mL (CoreRx lot number 111002-18011). The osmolality of this 12 mg / mL solution was 831 mOsm. For Venofer(R) Iron Sucrose Injection, 20 mg / mL, American Regent, lot number 8243A, the osmolality was 1742 mOsm. These osmolality measurements were performed without dilution.

[0038] [Example 4] The Western blot of the kidney 18 hours after administration of the aqueous iron composition is shown in Figure 2 and Table 7:

[0039] [Table 7]

[0040] The left side is a Western blot of the kidney's heavy chain specificity 18 hours after SnPP, FeS (Venofer), or Fe + SnPP. N = normal control. Glyc is glycerol and is used as a positive control for H-chain ferritin. N = normal sample (control). As is clear, Fe induces an increase in the heavy chain of the kidney.

[0041] [Example 5] Patients suffering from chronic kidney disease are treated by intravenous injection using the aqueous iron composition of iron sucrose and bicarbonate of Example 1.

[0042] [Example 6] Patients who have received an organ transplant are treated by intravenous injection using the aqueous iron composition of iron sucrose and bicarbonate of Example 1.

[0043] [Example 7] Patients who have received an organ transplant are treated by intravenous injection using the aqueous iron composition of iron sucrose and bicarbonate of Example 1 in combination with tin protoporphyrin.

[0044] [Example 8] Three samples of iron-sucrose (S1, S2) and iron-dextran (S3) were characterized by various analytical techniques. S1 was prepared according to Example 1 above. S2 is the commercial product Venofer® (iron sucrose injection). S3 is the commercial product INFeD® (iron dextran injection). The results are summarized in Table 8 below.

[0045] [Table 8] TIFF2025084949000011.tif215167TIFF2025084949000012.tif126167

[0046] Finally, the as-received sample S1 was titrated in triplicate with dilute HCl to determine the hydroxide value in the iron sucrose injection. The end point of the titration was pH = 7.0. Using the assumption that all the base species titrated were derived from the hydroxides associated with the ferric oxyhydroxide core, the total number of moles of H + was assumed to be equal to the number of moles of OH - . Considering the Mw (or Mn) by TOC and GPC, the molecular formula of iron sucrose in S1 was calculated as follows: Calculation based on Mw: [Na6Fe5O8(OH)5·3H2O] 13·73(C12H22O11) Calculation based on Mn: [Na6Fe5O8(OH)5·3H2O] 9·51(C12H22O11). Table 9 below shows the details of sample preparation and identification.

[0047]

Table 9

[0048] Sample preparation: Unless otherwise specified, the sample was lyophilized to a dry residue before analysis.

[0049] Gel permeation chromatography (GPC): GPC is used to determine the molecular weight distribution of polymers. In GPC analysis, a solution of the polymer is passed through a column filled with a porous gel. The sample is separated based on molecular size, and larger molecules elute faster than smaller molecules. The retention time of each component is detected, compared with a calibration curve, and then the data obtained are used to calculate the molecular weight distribution of the sample.

[0050] Rather than having a single molecular weight, it is characteristic of all types of synthetic polymers that the molecular weight is distributed. Statistical averages are used to characterize this distribution. The most common of these average values are the "number-average molecular weight" (Mn) and the "weight-average molecular weight" (Mw).

[0051] The number-average molecular weight is similar to the standard arithmetic mean related to a group of numbers. When applied to a polymer, the number-average molecular weight refers to the average molecular weight of the molecules in the polymer. The number-average molecular weight is considered to give the same amount of significance to each molecule regardless of its individual molecular weight. The number-average molecular weight is obtained by the following formula, where Ni is the number of molecules having a molar mass equal to Mi.

[0052]

Chem.

[0053] The weight-average molecular weight Mw is calculated somewhat differently and has a significantly different meaning. The weight-average molecular weight is another statistical descriptor of the molecular weight distribution and provides significance to relatively larger molecules compared to relatively smaller molecules in the distribution. The following formula shows the statistical calculation of the weight-average molecular weight.

[0054]

Chem.

[0055] For GPC, samples were prepared by diluting with phosphate buffer (by the USP monograph method) and analyzed to determine the molecular weight distribution of each sample. The results are summarized in Tables 10 - 12 below. Representative chromatograms from the analysis are shown in Figures 3 - 9.

[0056] There are two general reasons for the weight-average molecular weight. First, when comparing, for example, toughness, longer molecules have a greater impact on the toughness of the polymer distribution than shorter molecules. The weight-average molecular weight calculation places emphasis on these longer molecules and provides a measure that can account for the relative contribution of the longer molecules present in the molecular weight distribution. The weight-average molecular weight is also a number that directly correlates with the determination of the molecular weight of polymers by light scattering, small-angle neutron scattering (SANS), and sedimentation velocity.

[0057] Second, the weight-average molecular weight provides insight into the shape of the molecular weight distribution. This value determines the ratio of the spread of the molecular weight distribution, called the polydispersity index or PI, in relation to the number-average molecular weight. PI is defined as the ratio of Mw / Mn. The larger the PI, the more dispersed the distribution. The lowest value that PI can take is 1. This represents a monodisperse sample, i.e., a polymer in which all the molecules in the distribution have the same molecular weight.

[0058] Although not commonly referred to, the "z-average molecular weight" (Mz) can also be obtained. This molecular weight average is a value that further describes the molecular weight distribution. This value can be easily determined from sedimentation equilibrium.

[0059] The peak molecular weight Mp may also be included. The peak molecular weight value is taken as the mode of the molecular weight distribution. This means the most abundant molecular weight in the distribution. This value also provides insight into the molecular weight distribution.

[0060] Most GPC measurements are performed against different polymer standards (usually polystyrene). The accuracy of the results depends on how closely the characteristics of the polymer being analyzed match the characteristics of the standard being used. The expected error in reproducibility between different sets of separately calibrated measurements is about 5 - 10%, which is characteristic of the limited accuracy of GPC determinations. Therefore, GPC results are most useful when comparing the molecular weight distributions of different samples during the same set of measurements.

[0061] The accuracy and bias of GPC are based on statistical data such as the mean, standard deviation, relative percent difference, and / or percent relative standard deviation of the measured values. For quantitative analysis, the quantities listed in the above table are quantitative based on known amounts of reference substances. A calibration curve is created and information on relative standard deviation and relative percent difference is referenced in the above report. For the semi - quantitative typical reproducibility determined by statistical process control of the measurement system, it is estimated to be about 10% (95% confidence level, k is about 2). This reproducibility is an estimate of the uncertainty of a single standard measurement over time, and the uncertainty in a specific measurement must be determined on a case - by - case basis. For qualitative analysis, the analytical reference standards were not analyzed to confirm the presence of individual components. In such cases, it is not possible to assign a numerical value to the "uncertainty" of the provided match.

[0062] Note that sample S1 and S2 contained two peaks with different molecular weight distributions, while sample S3 contained three peaks. Also, it should be noted that for "Peak 2" (the small - molecule peak, probably sucrose), the peak saturated the detector, so Mp could not be calculated. The sample was analyzed at a concentration suitable for characterizing higher - molecular - weight species, sacrificing saturation of the detector with lower - molecular - weight species of less interest.

[0063]

Table 10

[0064]

Table 11

[0065]

Table 12

[0066] Dynamic light scattering PSD analysis was performed using a laser diffraction device. The measurement calculates the volume distribution from the laser diffraction pattern of the particle swarm. Next, this raw scattering data is processed with an algorithm and presented based on the equivalent spherical diameter. The results are summarized on a volume (mass) basis in a histogram giving the differential volume percentages smaller and larger than the indicated size.

[0067] Particle size analysis was performed using a Malvern(R) Zetasizer Nano ZS dynamic light scattering (DLS) device. DLS is an ensemble technique that analyzes light scattered by particles moving due to Brownian motion and generates a particle size distribution based on the diffusion rate of the particles. The raw scattering data is processed using a complex algorithm and presented based on the intensity-weighted HYDRODYNAMIC DIAMETER. This analytical technique is outlined in ISO22412:2008 Particle Size Analysis - Dynamic Light Scattering (DLS) as well as ASTM E2490-09(2015) Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy (PCS).

[0068] The as-received sample was water for injection (WFI), and the overall physical dimensions of the particles were obtained by analysis with DLS. The intensity and volume-weighted results from the analysis are shown in Tables 13 and 14, respectively.

[0069]

Table 13

[0070]

Table 14

[0071] Zeta potential Samples were prepared for zeta potential by diluting the sample with buffer (the device could not achieve stable readings when diluted with 10 mM NaCl around August 25, 2018 of the nanomaterial). The pH and temperature were recorded at the time of zeta potential analysis. The results are summarized in Tables 6 - 8 below. Stable readings could not be obtained for S2. The results of the zeta potential test are reported in Tables 15 - 17.

[0072]

Table 15

[0073]

Table 16

[0074]

Table 17

[0075] Atomic Force Microscope (AFM) The received samples were diluted 50 - fold using MilliQ filtered water (18.2 MΩ / cm, TOC 4 ppb). Approximately 10 μL of these diluted solutions were deposited onto freshly cleaved mica sheets and incubated for approximately 1 minute. The samples were then rinsed 5 times with MilliQ water and dried with nitrogen. Two 1 μm × 1 μm regions were imaged for each sample. The topographical differences in these images are represented by color, with brown being low and white being high. The z - range is shown by the vertical scale bar on the right side of the image. Perspective (3D) views of these surfaces are also included, with vertical exaggeration described in the caption.

[0076] Particle size analysis was performed to characterize the height of the particles present within each region. A height threshold of 0.5 nm was used to identify the particles of interest while excluding non - representative features. The results of the maximum height, minimum height, and average height are summarized in Table 18.

[0077]

Table 18

[0078] Cross-sectional analysis was performed, and the height of representative particles was measured manually. The cross-sectional analysis of S1 at position 1 is shown in FIGS. 10, 11, and 12. The results are summarized in Table 19 for each of S1, S2, and S3.

[0079]

Table 19

[0080] Total organic carbon (TOC) The total organic carbon (TOC) in the sample was calculated by subtracting the inorganic carbon from the total carbon (determined using a combustion carbon analyzer). The results are summarized in Table 20 below.

[0081]

Table 20

[0082] Osmolality The weight osmolality of the sample was measured using the vapor pressure method. The vapor pressure method determines the weight osmolality at room temperature using a sample in a natural equilibrium state. The results of the weight osmolality test are summarized in Table 21.

[0083]

Table 21

[0084] Fe +3 For Fe +2 An aliquot of each sample was diluted in concentrated hydrochloric acid according to the reference of the method provided by the client, Gupta et al. Subsequently, the sample was analyzed according to the method outlined by Stookey.2 The results are shown in Table 22.

[0085]

Table 22

[0086] Element screening by inductively coupled plasma / mass spectrometry (ICP / MS) and total iron and sodium contents by inductively coupled plasma / optical emission spectrometry (ICP / OES) ICP / OES is a spectroscopic technique used to identify and quantify components on an elemental basis. In ICP, inductive coupling transfers high-frequency energy to a stream of inert gas containing the sample as an aerosol. The energy vaporizes the aerosol while exciting the resulting free atoms to emit light. In this case, the intensity of this light is related to the concentration of the emitting atoms. This technique requires calibration of the instrument and calibration verification of a second source before, during, and after the analytical run sequence. Additionally, the instrument blank complies with each test verification specification. This ensures no carryover during the analysis sequence. Concentration measurements of major elements performed by ICP typically have an uncertainty in the range of 3 - 5% (at a 95% confidence level). The uncertainty in the concentration of trace elements can be significantly higher.

[0087] Samples S1 - S3 were analyzed by ICP-MS for metals and / or other elements. The samples were analyzed by ICP-OES to determine the total iron and sodium contents. The samples were analyzed while received in triplicate. The results are summarized in Tables 23 - 25.

[0088] [Table 23] TIFF2025084949000030.tif238163

[0089] [Table 24] TIFF2025084949000032.tif191164

[0090] [Table 25] TIFF2025084949000034.tif254162TIFF2025084949000035.tif148162

[0091] Fourier Transform Infrared Spectroscopy (FT-IR) Fourier Transform Infrared Spectroscopy (FT-IR) is the means selected for the identification of materials. In FT-IR, infrared absorption bands are assigned to characteristic functional groups. Based on the presence of several such bands, the material under consideration can be identified. The availability of spectra of known compounds increases the probability of making a positive identification. Lyophilized samples were analyzed by horizontal attenuated total reflection (HATR) based on the internal reflection of infrared radiation (IR). The FT-IR spectrum of S1 with a match in the spectral library is shown in Figure 13 below. The data suggests that the substance matches sucrose. The FT-IR spectra of S2 and S3 are shown in Figures 14 and 15. The assignments of absorption to functional groups are shown in Tables 26 to 28.

[0092]

Table 26

[0093]

Table 27

[0094]

Table 28

[0095] 1H Nuclear Magnetic Resonance Spectroscopy (NMR) NMR spectroscopy is a very useful method for the characterization of materials. NMR is a physical phenomenon based on the magnetic properties of atomic nuclei. NMR studies magnetic nuclei (most commonly those of hydrogen atoms) by aligning them with a very strong external magnetic field and perturbing this alignment using an electromagnetic pulse. The response to the perturbation is recorded, and each individual nucleus gives a response that is characteristic of its chemical, electronic, and spatial environment.

[0096] The lyophilized sample was reconstituted with heavy water (D2O) and analyzed by 1H NMR spectroscopy.

[0097]

Chem.

[0098] The structure of sucrose is shown above together with the hydrogen annotation of formula (I). The 1H NMR of S1 is shown in Table 29 below:

[0099]

Table 29

[0100]

Chem.

[0101] The structure of dextran is shown above using the hydrogen annotation of formula (II). Table 30 below shows the 1H NMR of S3.

[0102]

Table 30

[0103] The NMR spectra of the prepared samples are shown in Figures 16 - 18. Where possible, tentative assignments of the major chemical shifts observed in the NMR spectra were based on reference spectra of related compounds available in the literature.

[0104] The data indicate that sucrose is present in sample S1 and the chemical shifts are in good agreement with those reported in the literature. However, no peak splitting pattern was observed, which could be due to a number of reasons such as the presence of nanoparticles or paramagnetic iron itself.

[0105] The 1H NMR spectrum of sample S2 shows a significant amount of peak broadening. It is unclear whether this is due to the fine particles increasing the number of chemical environments or whether the nature of the iron in the sample could be causing the lack of resolution. Due to the degree of broadening, peak assignment could not be performed. However, a large broad response was observed from chemical shifts of 2.5 - 4.2 ppm, and a slight shoulder can be seen at the solvent peak around 5.5 ppm, so the general peak intensities and chemical shifts are consistent with those observed for sucrose.

[0106] 13C nuclear magnetic resonance spectroscopy (NMR) The lyophilized samples were reconstituted in deuterium oxide (D2O) and analyzed by 13C NMR spectroscopy.

[0107] The results are summarized in Tables 28 - 30. The NMR spectra of the prepared samples are shown in Figures 19 - 21. Where possible, tentative assignments of the major chemical shifts observed in the NMR spectra were based on reference spectra of related compounds available in the literature.

[0108] The data indicate that sucrose is present in samples S1 and S2 and that the chemical shifts are in good agreement with those reported in the literature. Note that, as with the proton spectra, sample S2 appeared to be more broadened than sample S1. Finally, the peaks observed in sample S3 are in good agreement with the literature values for dextran, indicating its presence in the sample.

[0109]

Chem.

[0110] The structure of sucrose is shown above using carbon annotation. The results of 13C NMR are shown in Table 31 below.

[0111]

Table 31

[0112]

Table 32

[0113]

Chem.

[0114] The structure of dextran is shown above with the carbon annotation of formula (III). The following Table 33 shows the 13C NMR of dextran of S3 in D2O:

[0115]

Table 33

[0116] X-ray diffraction (XRD) analysis (lyophilized product) XRD analysis is a method of irradiating a crystalline inorganic sample with monochromatic X-rays. The interaction between the lattice structure of the sample and these X-rays is recorded, providing information about the irradiated crystal structure. The resulting characteristic "fingerprint" enables the identification of the crystalline compounds present in the sample. Quantitative analysis can be performed on samples containing two or more crystalline compounds using whole pattern fitting analysis (Rietveld method).

[0117] The lyophilized samples were analyzed by XRD to characterize the chemical structures and phases present in the samples. The results of the analysis are shown in Table 34. Note that in this sample preparation method, the samples of S1 and S2 were particularly sticky (S3 had low stickiness). For S1 and S2, a drop of methanol was added to the sample and the material was spread flat in the sample holder. Sample S3 was ground with a mortar and pestle.

[0118]

Table 34

[0119] Figure 22 overlays the XRD raw data from three samples with a small offset for clarity. Sample S2 is different from the other two samples in terms of overall intensity, peak position, and peak shape. The broad peak shapes of Samples S1 and S3 indicate that these samples consist of a mixture of nanocrystalline and amorphous materials.

[0120] Using the best fit obtained by comparing the experimental data of the background model with the ICDD / ICSD diffraction databases of Samples S1, S2, and S3 respectively, Samples S1 and S3 were determined to contain a mixture of amorphous and nanocrystalline materials. The iron oxide sodium standard pattern was overlaid on these experimental data. The markers indicate the positions of the expected diffraction peaks of each phase, and the height of the markers indicates the relative peak intensities of the micronized randomly oriented materials. Different from the other two samples, Sample S3 is mainly composed of sucrose and amorphous materials.

[0121] Semi - quantitative analysis was performed using WPF (Whole Pattern Fitting), a subset of the Rietveld method that occupies all regions above the background curve. This technique requires that either the structure factor and atomic positions or the reference intensity ratio (a way to compare the diffraction power of different phases) be known for all identified phases. During this process, the structure factor (related to concentration), lattice parameters (related to peak position), peak width, and peak shape are refined for each phase to minimize the R - value, an estimate of the agreement between the model and the experimental data over the whole pattern.

[0122] To obtain quantitative results from samples containing measurable amounts of amorphous material, an amorphous density must be assigned to determine the amount of amorphous material present. As a result, the concentration of amorphous material is uncertain. The position of the amorphous peak in these samples is assumed to originate from amorphous sucrose, which has a density of approximately 1.59 g / cm3. Because the WPF attempts to account for everything in the sample, any error in the amorphous concentration will also result in an error in the crystalline phase. This means that the relative concentrations of the crystalline phases are correct, but the absolute values ​​will be in error by an amount proportional to the error in the amorphous concentration.

[0123] X-ray diffraction (XRD) analysis (sugar-free material) The as-received samples were diluted with water, placed in a 10,000 Da molecular weight cut-off (MWCO) filter, and centrifuged to remove small molecules (sugars) in the formulation, resulting in amorphous material for conventional XRD analysis. The samples were then washed an additional five times with water to remove residual small molecules. The resulting material (which could pass through the filter) was lyophilized and analyzed by XRD to characterize the chemical structure and phases present in the sample. Note that sample S3 contained two distinct layers after centrifugation, a thick viscous layer and a thin top layer. These layers were separated, lyophilized separately, and analyzed as two samples. The results were averaged to obtain the values ​​seen in Table 35, although individual replicate samples of each layer are shown in the figures below. The results of the analysis are shown in Table 35.

[0124] [Table 35]

[0125] An overlay of the XRD patterns from all four samples (two replicate samples for S3) is shown in Fig. 23. The patterns are offset for clarity. Phase identifications were made by comparing the best fit between the experimental XRD data of the background model and the ICDD / ICSD diffraction database of the samples. The phase reference markers indicate where the experimental peaks would be expected to be located at 2θ when the samples are micronized and randomly oriented, and the height of the markers indicates the expected intensity of the experimental peaks. Note that XRD is sensitive to the crystal structure but relatively unresponsive to the elemental or chemical state composition. The phase identifications of these samples were difficult due to the nanocrystallinity of the samples, which broadened the peaks of the XRD patterns significantly.

[0126] The peaks that match best in all four samples are an iron oxide phase and an iron oxyhydroxide phase known as maghemite. The iron oxyhydroxide phase is atypical because it is formed from heating of the beta-phase iron oxyhydroxide mineral up to about 300 °C. Unfortunately, the card for this standard does not include the reference intensity ratio (RIR) required for semi-quantitative analysis. However, since the symmetry and composition are the same as those of the iron oxyhydroxide mineral goethite (α-FeOOH), the average RIR of goethite was used for the iron oxyhydroxide for semi-quantitative analysis.

[0127] Semi-quantitative analysis was performed using WPF (whole pattern fitting), a subset of the Rietveld method that accounts for all intensities above the background curve. This technique requires that either the structure factors and atomic positions or the reference intensity ratios (a way to compare the diffraction power of different phases) are known for all identified phases. During this process, the structure factors (related to concentration), lattice parameters (related to peak position), peak widths, and peak shapes are refined for each phase to minimize the R-value, an estimate of the agreement between the model and the experimental data over the whole pattern.

[0128] Acid decomposition of labile iron(III) using ultraviolet-visible spectroscopy UV / Vis spectroscopy is used to determine analyte concentration either once or often over a desired period. This technique measures the absorption of light over ultraviolet and visible wavelengths through a liquid sample. The sample is dispensed into a small vial and placed between the path of the UV / Vis light and the detector. According to Beer-Lambert's law, using a known absorption coefficient that depends on a constant optical path length and wavelength, the concentration of the compound in question can be determined from the light absorbed by the sample at that wavelength.

[0129] Samples were analyzed using a method adapted from BS. Barot et al. (2014) that uses UV-visible spectroscopy to determine the amount of unstable iron(III) in the sample. The results are summarized in Table 36 below.

[0130] [Table 36]

[0131] Thermogravimetric analysis (TGA) TGA consists of measuring the weight change of a material as a function of temperature in a controlled atmosphere. This technique requires accurate measurement of weight, temperature, and temperature change. The resulting thermogram generated from the analysis can determine the content of component classes (e.g., solvents, polymers, inorganic fillers, etc.) and the thermal stability of the polymer. The typical accuracy and bias for TGA measurements are discussed in ASTM E2040.

[0132] Lyophilized samples were analyzed by thermogravimetric analysis (TGA) under nitrogen purge and air purge. The thermal decomposition of the samples occurs in three different steps, as shown in Figure 24. The results of these steps are summarized in Table 37.

[0133] [Table 37]

[0134] Differential scanning calorimetry (DSC) and differential thermal analysis (DTA) The lyophilized sample was analyzed by differential scanning calorimetry (DSC) under argon purge. Differential scanning calorimetry (DSC) measures the difference in heat flow associated with transitions between a sample and an inert reference as a function of temperature and time. Such measurements provide quantitative and qualitative information regarding endothermic or exothermic processes, or physical and chemical changes involving changes in heat capacity. See Figure 25 for the DSC thermogram. An overview of DTA is shown in Table 38 below.

[0135] [Table 38]

[0136] Determination of Hydroxide Value and Molecular Formula by Titration The as-received sample S1 was titrated in triplicate with 0.00998 N HCl to determine the hydroxide value in the iron sucrose injection. The endpoint of the titration was pH = 7.0. Table 39 summarizes the results of this titration for S1.

[0137] Assuming that all the base species titrated are derived from the hydroxide associated with the ferric oxyhydroxide core, it was assumed that the total number of moles of H+ used in the titration is equal to the number of moles of OH-. Considering Mw by TOC and GPC, the molecular formula of iron sucrose in S1 was calculated as follows: [Na6Fe5O8(OH)5·3H2O]13·73(C12H22O11) Considering Mn for this calculation, the molecular formula is as follows.

[0138] [Na6Fe5O8(OH)5·3H2O] 9·51(C12H22O11)

[0139] [Table 39]

[0140] Other embodiments and uses of the invention will be apparent to those skilled in the art from a consideration of this specification and practice of the invention disclosed herein. All references cited herein, including all U.S. and foreign patents and patent applications, are hereby incorporated by reference in their entirety and specifically. This specification and examples are considered to be merely illustrative, and it is intended that the true scope and spirit of the invention be indicated by the following claims.

Claims

1. 1. An aqueous iron composition comprising: Iron sucrose; and bicarbonate Including, 1. An aqueous iron composition, wherein the composition has a pH greater than 9.

2. 10. The aqueous iron composition of claim 1, wherein the composition has a pH in the range of 10.5 to 11.

5.

3. 10. The aqueous iron composition of claim 1, wherein the composition has a pH in the range of 10.1 to 10.

4.

4. 4. The aqueous iron composition of claim 1, wherein the composition has a specific gravity of 1.135 to 1.165 at 20°C.

5. 5. The aqueous iron composition of any one of claims 1 to 4, wherein the composition has a Mw by GPC of 30,000 to 40,000 Daltons.

6. 6. The aqueous iron composition of claim 5, wherein the composition has a Mw by GPC of 33,000 to 38,000 Daltons.

7. 7. The aqueous iron composition of any one of claims 1 to 6, wherein the composition has a maximum concentration of iron(II) of 0.40% w / v.

8. 8. The aqueous iron composition of claim 7, wherein the composition has a concentration of iron(II) of 0.05% w / v to 0.40% w / v.

9. 9. The aqueous iron composition of claim 8, wherein the composition has a concentration of iron(II) of 0.10% w / v to 0.20% w / v.

10. 10. The aqueous iron composition of claim 1, further comprising a pharmaceutically acceptable aqueous carrier, wherein the iron sucrose is present in a pharmaceutically effective amount to provide a protective effect to the patient's kidneys, has an iron(II) concentration of 0.05% w / v to 0.40% w / v, and has a Mw by GPC of 30,000 to 40,000 Daltons.

11. 11. The aqueous iron composition of claim 10, wherein the composition has a pH in the range of 10.5 to 11.

5.

12. 11. The aqueous iron composition of claim 10, wherein the composition has a pH in the range of 10.1 to 10.

4.

13. 13. The aqueous iron composition of any one of claims 10 to 12, wherein the composition has a specific gravity of 1.135 to 1.165 at 20°C.

14. 14. The aqueous iron composition of any one of claims 10 to 13, having a Mw by GPC of 33,000 to 38,000 Daltons.

15. 15. The aqueous iron composition of any one of claims 10 to 14, wherein the composition has a concentration of iron(II) of 0.10% w / v to 0.20% w / v.

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